Primary sequence of a dimeric bacterial haemoglobin from Vitreoscilla

Vitreoscilla, a filamentous bacterium in the Beggiatoa family, synthesizes a soluble haem-protein which has two identical subunits of relative molecular mass 15,775 and two b haems per molecule. It is synthesized in relatively large quantities when the organism, a strict aerobe, is grown under hypoxic conditions. It forms a relatively stable oxygenated form which is spectrally similar to oxymyoglobin (oxyHb) and oxyhaemoglobin (oxyHb). The amino acid sequence of this protein has been determined and aligned to fit the helical regions of several animal and plant globins. This alignment is consistent with its being a structural homologue of the eucaryotic haemoglobins although it diverged from the others in the N-terminal region and may lack an A-helix. It showed the maximum sequence homology (24%) with lupin leghaemoglobin (Lb). Vitreoscilla Hb is the first bacterial haemoglobin to be sequenced. It may function to enable the organism to survive in oxygen-limited environments by acting as an oxygen storage-trap or to facilitate oxygen diffusion.     

Extended X-ray absorption fine structure determination of iron nitrogen distances in haemoglobin

EXAFS spectra have been obtained of oxy and deoxy complexes of haemoglobin and of the 'picket fence' porphyrin, using synchrotron radiation as a source of X rays. The fluorescence data were Fourier filtered to obtain distances to the first shell and corrections applied to remove contributions from the axial ligands. In this way, the iron to porphinato nitrogen distances were determined to be 1.98 +/- 0.01 A for both oxygenated complexes and 2.055 +/- 0.01 A for both deoxy forms.     

A haemoglobin switching activity modulates hereditary persistence of fetal haemoglobin

During human development there is a switch from fetal to adult haemoglobin formation, reflecting the differential expression of fetal (G gamma and A gamma) and adult (beta and delta) globin genes. Mutations that inhibit this switch produce variants of the syndrome of hereditary persistence of fetal haemoglobin (HPFH). Adult heterozygotes for these mutants produce 15-30% fetal haemoglobin (HbF) in their red cells. The general assumption is that the mutations result in a permanent switching on of gamma-globin genes. Here, however, we show that fetal globin expression can be turned off in cultures of HPFH cells by an uncharacterized factor in fetal sheep serum. This is the first demonstration that mutations affecting the developmental expression of globin genes can be modulated by exogenous factors. The findings raise the possibility that the phenotype of HPFH is not simply the direct result of mutations in or around globin genes but the consequence of the mutations on the interaction of globin genes with trans-acting regulatory factors.     

Primary structure of human T-cell receptor alpha-chain

The T-cell receptor has been studied intensely over the past 10 years in an effort to understand the molecular basis for major histocompatibility complex (MHC) restricted antigen recognition. The use of anti-receptor monoclonal antibodies to isolate and characterize the receptor from human and murine T-cell clones has shown that the protein consists of two disulphide-linked glycopeptides, alpha and beta, distinct from known immunoglobulin light and heavy chains. Like immunoglobulin light and heavy chains, however, both the alpha- and beta-chains are composed of variable and constant regions. Molecular cloning has revealed that the beta-chain is evolutionarily related to immunoglobulins, and is encoded in separate V (variable), D (diversity), J (joining) and C (constant) segments that are rearranged in T cells to produce a functional gene. We report here cDNA clones encoding the alpha-chain of the receptor of the human T-cell leukaemia line HPB-MLT. Using these cDNA probes, we find that expression of alpha-chain mRNA and rearrangement of an alpha-chain V-gene segment occur only in T cells. The protein sequence predicted by these cDNAs is homologous to T-cell receptor beta-chains and to immunoglobulin heavy and light chains, particularly in the V and J segments.     

Crystals of haemoglobin with the T quaternary structure bind oxygen noncooperatively with no Bohr effect

The relationship between the structure and function of haemoglobin has mainly been studied by comparing its X-ray crystal structures with its function in solutions. To make a direct comparison we have studied the functional properties of haemoglobin in single crystals, an approach that has been an important part of the investigation of several enzyme mechanisms. Here we report on the oxygen binding by single crystals of human haemoglobin grown in solutions of polyethylene glycol. Unlike haemoglobin crystals formed in concentrated salt solution, which crack and become disordered on oxygenation, crystals grown in polyethylene glycol remain intact. X-ray studies have shown that the T (deoxy) quaternary structure of haemoglobin in this crystal at pH 7.0 is maintained at atmospheric oxygen pressure, and that the salt-bridges are not broken. We find striking differences between oxygen binding by haemoglobin in this crystal and by haemoglobin in solution. Not only is oxygenation of the crystal noncooperative, but the oxygen affinity is independent of pH in the range 6.0-8.5, and is much lower than that of the T state in solution. The lack of cooperativity without a change in quaternary structure is predicted by the two-state allosteric model of Monod, Wyman and Changeux. The absence of a Bohr effect without breakage of salt-bridges is predicted by Perutz's stereochemical mechanism. In contrast to the X-ray result that oxygen binds only to the alpha haems, our measurements show that the alpha haems have only a slightly higher affinity than the beta haems.